PATHOGENESIS AND VIRULENCE FACTORS Influence of Different Levels of Redox Potential on Fermentative Products Formed by Bacteroides fragilis. M. GOLDNER, N. MINGOT,

The oxidation-reduction (redox) potential (Eh) of a system deence shown by variation of the redox potential, the degree of difference could be explored at an initial moderating level for rives from the measurement of transfer of electrons between a pair of electrodes in relation to the standard hydrogen electrode. The comparison with oxidizing or reducing levels. The accumulation or depletion of certain influential components and the influence of Eh of culture medium can be measured on a scale expressed in millivolts (mV) by use of a platinum electrode connected through these fermentative products at particular levels of redox potential on formation of other products of this type could manifest a change a voltmeter to a calomel reference electrode with known Eh. Bacteroides fragilis can be grown in a chemically defined mein character of this potential pathogen. A change in character of the potential pathogen relative to the level of redox potential might dium containing cysteine (0.5 g/L) as reducing agent [1, 2]. This potential at the platinum electrode depends on the proportions of reasonably suggest some form of redox switching mechanism. This means that the microorganism, be it in the commensal or the oxidized and reduced forms of the added cysteine. The more reduced, the lower the Eh will be, i.e., the more negative on the pathogenic state, will have more, or less, accessibility to the pathways in carbohydrate catabolism. redox scale (and vice-versa, the more oxidized the added substance is). A special combination platinum-calomel electrode (XM-800; The effect of each of pyruvic, acetic, and lactic acids—constitutTacussel, Lyon, France) would be used for Eh (adjusted by calculaing the anaerobe/aerobe metabolic interface in the pathway of tion for Eh at pH7), and a classic combination glass-Ag/AgCl glucose degradation in B. fragilis [5]—on formation of other comelectrode (TC 100; Tacussel) would be used for pH. ponents of this type was analyzed after growth under the influence In previous studies, these bacteria have been grown at three of these different levels of redox potential. The amounts of each initial redox conditions. The amount of cysteine was adjusted to acid added were estimates determined from their formation in 480.05, 0.25, and 2.5 g/L [3], which provided readings of Eh7 [4] hour culture under usual conditions of growth (0.5 g/L of cysteine) at, respectively, circa (ca) /100 mV for oxidizing, ca /20 mV [1–3]. The amount of cysteine in the chemically defined medium for moderating, and ca 060 mV for reducing conditions. By vary(L-cysteine hydrochloride hydrate, Aldrich Chemical, Milwaukee) ing the levels of redox potential at start of growth, the studies was adjusted as previously described to 0.05, 0.25, and 2.5 g/L revealed potential pathogenic characteristics of B. fragilis [3] [3]. The B. fragilis Institut Pasteur (IP) 5-86 clinical isolate was grown until mid-log phase in the defined medium [1, 2]. A strong used and conditioned (pH, 7; 377C) in 10 mL of defined medium penetration into HeLa cells was observed with bacteria grown at [1, 2], containing a concentration of cysteine (0.5 g/L) in sealinitial oxidizing conditions (Eh7 ca /100 mV); a weak penetration capped, rubber-stoppered serum bottles (95% H2, 5% CO2). occurred with bacteria grown at moderating conditions (Eh7 ca For the redox experiments, the bacterial inoculum (0.05 mL), /20 mV). A compact aggregation at the HeLa cell surface was obtained after usual conditioning (2–4 overnight subcultures), was formed with bacteria grown at reducing conditions (Eh7 ca 060 inoculated into cultures that were then grown in this defined memV). dium, except that the concentrations had been adjusted to the oxiThis sharp distinction in terms of the level of redox potential dizing, moderating, and reducing conditions. It has been shown observed in the interaction between B. fragilis and HeLa cells has that in the defined medium [1, 2] members of the Bacteroides revealed a striking adaptation with a pathogenic quality to a change genus have only limited use of amino acids; thus, use of cysteine in environment. Mucous material appeared on the bacteria after as a metabolite was probably only of minor importance, and this growth at initial reducing conditions (Eh7 ca 060 mV); this may should be beneficial in achieving Eh7 [4]. mask receptor sites on the tissue cells and therefore hinder an The formation of fermentative products by B. fragilis IP 5-86 invasive process [3]. Thus, these previous studies have shown that after growth at the different levels of redox potential was deterthe pathogenic potential of B. fragilis may be reflected in the redox mined on the 48-hour culture supernatants analyzed by gas chroconditions. matography (Hewlett Packard [Detroit] 5890 Series II, Supelco A conceptual argument could be put forward with regard to FFAP column, 10 m length by 0.53 mm internal diameter; temperametabolic change and pathogenic potential. Because of the influtures: detector [flame ionization detector], 2007C, programmed from 907C to 1307C, with progression at 57C/min; extracts prepared according to VPI [Blacksburg, VA] system; sample injection, 2 mL). The percent increase in formation of metabolic components The authors express their sincere appreciation to Christiane Tigoulet, for (those distributed through the pathway of glucose degradation) her expert analyses by gas chromatography, and to Michel Picard, for his devoted attention to preparation of materials. upon addition of the components constituting the anaerobe/aerobe Reprints or correspondence: Dr. Morris Goldner, Département de Microbiinterface (pyruvic, acetic, and lactic acids) was calculated over ologie, Faculté de Médecine, Université Laval, Ste-Foy (Québec), Canada G1K that of no addition for base-unit growth in each instance under the 7P4. varying redox conditions (oxidizing, moderating, and reducing). Clinical Infectious Diseases 1997;25(Suppl 2):S147–50 The addition of lactic acid at oxidizing conditions, in contrast q 1997 by The University of Chicago. All rights reserved. 1058–4838/97/2503–0019$03.00 with that at moderating and reducing conditions, resulted in a